ABSTRACT Raman spectroscopy is widely employed for quantitative analysis in aqueous solutions, yet it faces a notable challenge: the overlap of water Raman bands, often used as an internal standard, with analyte bands. To overcome this hurdle, we have developed an automated fitting method that encompasses intensity normalization, solvent subtraction, and quantitative analysis. This method utilizes the isolated Raman water spectrum as an internal standard. The effectiveness of this method has been validated through its application to the Raman spectra of Na 2 SO 4 , D‐glucose, and lysozyme from egg white. These results show a strong correlation between normalized peak intensity and concentration. Furthermore, this method exhibits robustness against noise and fluorescence background, maintaining high accuracy even under low–signal‐to‐noise ratio (SNR) conditions, down to 20 dB. Most importantly, the fundamental principle of this method is versatile and can be applied to various types of quantitative spectral data derived from solutions.